Biophysical analysis of the endoplasmic reticulum-resident chaperone/heat shock protein gp96/GRP94 and its complex with peptide antigen.

Animals vaccinated with heat shock protein (HSP)--peptide complexes develop specific protective immunity against cancers from which the HSPs were originally isolated. This autologous specific immunity has been demonstrated using a number of HSP--peptide antigen complexes. A prototypical HSP-based cancer vaccine is the gp96--peptide antigen complex, which is currently undergoing human clinical trials. Here, we analyzed the structure of a recombinant wild-type and a mutant gp96 protein and their peptide complexes using a number of biophysical techniques. Gel filtration chromatography, dynamic light scattering, and equilibrium analytical ultracentrifugation demonstrated that both a wild-type gp96 and a gp96 mutant lacking a dimerization domain formed higher order structures. More detailed analysis using scanning transmission electron microscopy indicated that both the wild-type and dimerization deletion mutant gp96 protein were organized, unexpectedly, into large aggregates. Size distributions ranged from dimers to octamers and higher. Circular dichroism and intrinsic Trp fluorescence suggested that the gp96 dimerization domain deletion mutant protein was more compact than the wild-type gp96. A fluorescent peptide antigen was synthesized, and the peptide-binding properties of wild-type and the dimerization domain deletion mutant gp96 were studied. Fluorescence lifetime and anisotropy decay showed that the bound antigenic peptide was located in a hydrophobic pocket, with considerable free space for the rotation of the probe. Deletion of the dimerization domain affected the peptide-binding microenvironment, although peptide-binding affinity was reduced by only a small extent. Peptide--gp96 complexes were extremely stable, persisting for many days in the cold. The extraordinary stability of peptide--gp96 complexes and the plasticity of the peptide-binding pocket support the proposed relay of diverse peptides to MHC and/or other molecules via molecular recognition.

Binding of antigenic peptide to the endoplasmic reticulum-resident protein gp96/GRP94 heat shock chaperone occurs in higher order complexes. Essential role of some aromatic amino acid residues in the peptide-binding site.

Vaccination with heat shock protein gp96-antigenic peptide complexes produces a powerful specific immune response against cancers and infectious diseases in some experimental animal models, and gp96-peptide complexes are now being tested in human clinical trials. gp96 appears to serve as a natural adjuvant for chaperoning antigenic peptides into the immune surveillance pathways. A fundamental issue that needs to be addressed is the mechanism of binding of antigenic peptide to gp96. Here, we show using scanning transmission electron microscopy that recombinant gp96 binds peptide in stable multimeric complexes, which may have biological significance. To open the possibility for genetically engineering gp96 for improved immunogenicity and to understand if molecular recognition plays a role in the binding of antigenic peptide, we mutagenized some specific aromatic amino acids in the presumed peptide-binding pocket. Replacement of Tyr-667 or Tyr-678 to Ala reduced affinity for peptide whereas conversion of Trp-654 to Tyr increased peptide binding. Similarly, changing Trp-621 to Phe or Leu or Ala or Ile negatively affected peptide binding whereas changing Trp-621 to Tyr or Val positively affected peptide binding. Probing the peptide microenvironment in gp96-peptide complexes, suggested that hydrophobic interactions (and perhaps hydrogen bonding/stacking interactions) may play a role in peptide loading by gp96.

Identification of the peptide-binding site in the heat shock chaperone/tumor rejection antigen gp96 (Grp94).

Heat shock protein (HSP)-peptide complexes from tumor cells elicit specific protective immunity when injected into inbred mice bearing the same specific type of tumor. The HSP-mediated specific immunogenicity also occurs with virus-infected cells. The immune response is solely due to endogenous peptides noncovalently bound to HSP. A vesicular stomatitis virus capsid-derived peptide ligand bearing a photoreactive azido group was specifically bound by and cross-linked to murine HSP glycoprotein (gp) 96. The peptide-binding site was mapped by specific proteolysis of the cross-links followed by analysis of the cross-linked peptides using a judicious combination of SDS-gel electrophoresis, mass spectrometry, and amino acid sequencing. The minimal peptide-binding site was mapped to amino acid residues 624-630 in a highly conserved region of gp96. A model of the peptide binding pocket of gp96 was constructed based on the known crystallographic structure of major histocompatibility complex class I molecule bound to a similar peptide. The gp96-peptide model predicts that the peptide ligand is held in a groove formed by alpha-helices and lies on a surface consisting of antiparallel beta-sheets. Interestingly, in this model, the peptide binding pocket abuts the dimerization domain of gp96, which may have implications for the extraordinary stability of peptide-gp96 complexes, and for the faithful relay of peptides to major histocompatibility complex class I molecule for antigen presentation.

The filamentous phage pIV multimer visualized by scanning transmission electron microscopy.

A family of homomultimeric outer-membrane proteins termed secretins mediates the secretion of large macromolecules such as enzymes and filamentous bacteriophages across bacterial outer membranes to the extracellular milieu. The secretin encoded by filamentous phage f1 was purified. Mass determination of individual molecules by scanning transmission electron microscopy revealed two forms, a unit multimer composed of about 14 subunits and a multimer dimer. The secretin is roughly cylindrical and has an internal diameter of about 80 angstroms, which is large enough to accommodate filamentous phage (diameter of 65 angstroms).

Filamentous phage assembly: variation on a protein export theme.

Biogenesis of both filamentous phage and type-IV pili involves the assembly of many copies of a small, integral inner membrane protein (the phage major coat protein or pilin) into a helical, tubular array that passes through the outer membrane. The occurrence of related proteins required for assembly and export in both systems suggests that there may be similarities at the mechanistic level as well. This report summarizes the properties of filamentous phage and the proteins required for their assembly, with particular emphasis on features they may share with bacterial protein export and pilus biogenesis systems, and it presents evidence that supports the hypothesis that one of the phage proteins functions as an outer membrane export channel.

In vivo and in vitro evidence for an anti-Rho activity induced by the phage P4 polarity suppressor protein Psu.

The polarity suppression (Psu) protein of bacteriophage P4 causes suppression of transcriptional polarity in Escherichia coli by overcoming Rho termination factor activity. Two new psu mutants defective in polarity suppression are described. The psu5 mutation deletes codons 95-98 from about the middle of the gene, and the mutant protein is inactive. The psu6 mutation changes Phe169 to Val and encodes a temperature-sensitive protein. Constitutive overexpression of psu+ from a plasmid prevents colony formation, but overexpression of mutant genes (psu5, psu6) does not, suggesting that Psu disturbs essential host function(s). Rho protein synthesis is enhanced several-fold in cells containing wild-type Psu, due to readthrough at the rho attenuator, while the physical stability of Rho is maintained. As a consequence, Psu-producing cells accumulate significantly more Rho than normal cells, reminiscent of termination-defective rho mutants. The polarity suppression activity induced by Psu is demonstrated in vitro by the efficient readthrough of Rho-dependent terminators lambda tR1 and TIS2 during coupled transcription-translation. Purified Rho protein restores termination at TIS2 when added to Psu-containing reactions but NusG does not. The data support the hypothesis that Psu has or elicits an anti-Rho function.

Essential role of a sodium dodecyl sulfate-resistant protein IV multimer in assembly-export of filamentous phage.

Filamentous phage f1 encodes protein IV (pIV), a protein essential for phage morphogenesis that localizes to the outer membrane of Escherichia coli, where it is found as a multimer of 10 to 12 subunits. Introduction of internal His or Strep affinity tags at different sites in pIV interfered with its function to a variable extent. A spontaneous second-site suppressor mutation in gene IV allowed several different insertion mutants to function. The identical mutation was also isolated as a suppressor of a multimerization-defective missense mutation. A high-molecular-mass pIV species is the predominant form of pIV present in cells. This species is stable in 4% sodium dodecyl sulfate at temperatures up to 65 degrees C and is largely preserved at 100 degrees C in Laemmli protein sample buffer containing 4% sodium dodecyl sulfate. The suppressor mutation makes the high-molecular-mass form of wild-type pIV extremely resistant to dissociation, and it stabilizes the high-molecular-mass form of several mutant pIV proteins to extents that correlate with their level of function. Mixed multimers of pIV(f1) and pIV(Ike) also remain associated during heating in sodium dodecyl sulfate-containing buffers. Thus, sodium dodecyl sulfate- and heat-resistant high-molecular-mass pIV is derived from pIV multimer and reflects the physiologically relevant form of the protein essential for assembly-export.

Domain structure of phage P4 alpha protein deduced by mutational analysis.

Bacteriophage P4 DNA replication depends on the product of the alpha gene, which has origin recognition ability, DNA helicase activity, and DNA primase activity. One temperature-sensitive and four amber mutations that eliminate DNA replication in vivo were sequenced and located in the alpha gene. Sequence analysis of the entire gene predicted a domain structure for the alpha polypeptide chain (777 amino acid residues, M(r) 84,900), with the N terminus providing the catalytic activity for the primase and the middle part providing that for the helicase/nucleoside triphosphatase. This model was confirmed experimentally in vivo and in vitro. In addition, the ori DNA recognition ability was found to be associated with the C-terminal third of the alpha polypeptide chain. The type A nucleotide-binding site is required for P4 replication in vivo, as shown for alpha mutations at G-506 and K-507. In the absence of an active DnaG protein, the primase function is also essential for P4 replication. Primase-null and helicase-null mutants retain the two remaining activities functionally in vitro and in vivo. The latter was demonstrated by trans complementation studies, indicating the assembly of active P4 replisomes by a primase-null and a helicase-null mutant.

Molecular cloning and characterization of bacteriophage P2 genes R and S involved in tail completion.

The sequences of two previously known tail genes, R and S, of the temperate bacteriophage P2 and the sequence of an additional open reading frame (orf-30) located between S and V, were determined. Amber mutations mapping within R and S, Ram3, Ram42, Ram23, Sam75, and Sam89 were sequenced and found to be within their corresponding open reading frames. We constructed overproducing plasmids for R and S and identified these proteins by SDS-PAGE of whole-cell lysates and Coomassie blue staining. The predicted molecular masses of proteins R and S were M(r) 17,400 and 17,300, respectively, although both polypeptides migrated more slowly during gel electrophoresis than would be expected from the sequence data. orf-30 occupies the strand opposite from RS and V and is preceded by several weak potential sigma 70-RNA polymerase promoters, some of which overlap with the V promoter. A construct that had the putative orf-30 promoter region upstream of the lacZ gene produced low levels of beta-galactosidase activity in vivo. Expression from the orf-30 promoter was not stimulated by the phage P4 transcriptional activator protein, delta, which acts at all the known P2 and P4 late promoters. Insertion mutagenesis showed that orf-30 was not an essential gene for P2 growth in Escherichia coli. None of the gene or protein sequences exhibited extensive homology to sequences in the nucleic acid and protein databases. However, the R protein contains a small region homologous to one in the phage T4 tail protein gp15, which is required for T4 tails to bind heads. We propose that R and S are tail completion proteins that are essential for stable head joining.

Nucleotide sequence of the DNA packaging and capsid synthesis genes of bacteriophage P2.

Overlapping DNA fragments containing the DNA packaging and capsid synthesis gene region of bacteriophage P2 were cloned and sequenced. In this report we present the complete nucleotide sequence of this 6550 bp region. Each of six open reading frames found in the interval was assigned to one of the essential genes (Q, P, O, N, M and L) by correlating genetic, physical and mutational data with DNA and protein sequence information. Polypeptides predicted were: a capsid completion protein, gpL; the major capsid precursor, gpN; the presumed capsid scaffolding protein; gpO; the ATPase and proposed endonuclease subunits of terminase, gpP and gpM, respectively; and a candidate for the portal protein, gpQ. These gene and protein sequences exhibited no homology to analogous genes or proteins of other bacteriophages. Expression of gene Q in E. coli from a plasmid caused production of a Mr 39,000 Da protein that restored Qam34 growth. This sequence analysis found only genes previously known from analysis of conditional-lethal mutations. No new capsid genes were found.

The Psu protein of bacteriophage P4 is an antitermination factor for rho-dependent transcription termination.

A 0.7-kbp DNA fragment from bacteriophage P4 that contained the polarity suppression (psu) gene was cloned in an expression plasmid. Induction of the plasmid-borne psu gene resulted in the overproduction of a protein having the biological properties of the P4-induced polarity suppressor. In vivo, Psu protein acted in trans to suppress rho-dependent polarity in the late genes of an infecting P2 phage, in plasmid operons, and in the host chromosome. Psu action did not require the presence of other P2 or P4 phage genes. Psu caused efficient readthrough (antitermination) by Escherichia coli RNA polymerase at the rho-dependent terminators tR1 and TIS2, individually and in tandem, but did not affect termination at rho-independent sites. Neither the conserved antitermination sequence boxA nor any unique promoter or utilization sequence was required for Psu activity.